The overall aim of the proposed work is to eludidate mechanisms of post transcriptional gene control focusing on the formation of MRNA poly(A) sites. The generation of the polyadenylated messenger RNA 3' end is clearly a crucial event in mRNA biogenesis and several studies demonstrate that it can contribute to the regulation of gene expression. Work in our laboratory, supported by this grant over the past five years, has been successful in identifying sequences in polyadenylation sites that are important for the processing event as well as identifying regulatory pathways involving poly(A) site utilization. More recently, we have used HeLa cell nuclear extracts to isolate factors that support authentic polyadenylation in vitro in a reconstituted system. The analysis of the interaction of these factors with the pre-mRNA has led to an understanding of the relative contributions of the factors in poly(A) site selection, and has provided insight into the manner in which the assembly of factors on an RNA contributes to poly(A) site utilization. We propose to continue these studies with a major focus on the biochemical analysis of polyadenylation factors that have been isolated and purified from HeLa cell nuclear extracts. We propose to complete the purification of these factors to allow detailed biochemical characterizations of the interactions of the factors with the processing complex as well as with the pre-mRNA. Antibodies will be produced against the various activities and genes encoding the polypeptide constituents of the activities will be cloned. We will also begin to explore the role of these factors in the regulation of poly(A) site utilization. These studies will focus on the utilization of the mu immunoglobulin poly A sites and their regulation during B cell differentiation.